The invention relates broadly to production of capsular polysaccharide material from streptococcal organisms and to production of vaccines using such material.
Streptococcus pneumoniae, sometimes designated pneumococcus, is generally a commensal organism colonizing the mucosal surface of the human nasopharynx. When host factors allow the organism access to the lower respiratory tract, a vigorous inflammatory response ensues, leading to a dense consolidation as alveolar air spaces fill with exudate. This condition is commonly referred to as pneumonia (Tuomanen et al., 1995). The most serious manifestation of pneumococcal infection is bacteremia, which can be complicated by sepsis, meningitis, or both. Bacteremia in adults is usually a complication of pneumonia (Raz et al., 1997).
The ability of the pneumococcus to resist the major mechanism of clearance of the organism from the bloodstream (i.e. opsonophagocytosis) requires expression of the major virulence factor of the organism, which is a polysaccharide capsule (Avery et al., 1931; Watson et al., 1990). Pneumococci are capable of synthesizing no fewer than 90 structurally unique capsular polysaccharides (CPSs).
Pneumococcal CPS exhibits anti-phagocytic properties and inhibits adherence to host cells, a critical step in carriage (i.e. spread of the organism from one infected, but not necessarily symptomatic, individual to another) and possibly later aspects in the pathogenesis of disease (Ring et al., 1998). Similar findings in other encapsulated species (e.g. Haemophilus influenzae, Neisseria meningitidis, Streptococcus pyogenes, and Streptococcus agalactiae) has led to an appreciation of how amounts of CPS must be temporally varied to allow both adhesive interactions with host cells and resistance to humoral immunity (Hammerschmidt et al., 1996; Levin et al., 1998; Schrager et al., 1996; Sellin et al., 1995; St. Geme III et al., 1991).
Even within a single strain, the amount of CPS expressed by S. pneumoniae varies. The regulatory mechanism(s) which modulate CPS expression have not previously been well understood. Most pneumococcal isolates undergo phenotypic variation between at least two forms, which can be distinguished by colony opacity (Weiser, 1998; Weiser et al., 1994). Opaque (O) colony forms differ from transparent (T) variants of the same strain in the amount of CPS that is synthesized, O-form colonies generally making a greater amount of CPS (Kim et al., 1998).
Relatively minor differences in the amount of CPS made by a pneumococcal variant can have a major impact on virulence (MacLeod et al., 1950). The 1.2 to 5.6-fold higher quantities of CPS produced by O-form colonies, compared to T-form colonies, correlates with increased resistance of pneumococcal cells to opsonophagocytosis using immune serum (Kim et al., 1999). In a murine model of systemic infection, only the O variant of several pneumococcal strains caused sepsis (Kim et al., 1998). In contrast, the T form expresses higher amounts of the other cell surface polysaccharide, teichoic acid. Pneumococcal cell-wall teichoic acid is covalently linked to CPS and contains an unusual host-like constituent, phosphorylcholine. This polysaccharide contributes to adherence of pneumococcal cells to epithelial cells via the receptor for platelet activating factor (Cundell et al., 1995; Cundell et al., 1995; Kim et al., 1998). The T form also exhibits an altered distribution of cell surface choline-binding proteins, including CbpA, which acts to promote adherence and colonization (Rosenow et al., 1997). During carriage in an infant rat model, there is selection for pneumococcal variants which exhibit the T, rather than O, phenotype (Weiser et al., 1994). These observations suggests that pneumococci vary between a form (i.e. the T-form) which exhibits greater adherence and carriage, and a non-adherent form (i.e. the O-form) which is better adapted to survival in invasive infection.
Identification of the CPSs expressed by a pneumococcus forms the basis of serotyping, a diagnostic procedure used for differentiating S. pneumoniae variants. Because different variants can exhibit different physiological properties (e.g. susceptibility to anti-microbial agents or characteristic rate of onset or progression of pneumonia), ability to differentiate pneumococcal variants is medically advantageous. Furthermore, because the ability of the human (or other vertebrate) immune system to recognize and attack pneumococcal variants depends on its ability to specifically recognize the CPS of each variant, ability to obtain pneumococcal variant-specific CPS significantly affects development of therapeutic and preventive methods and compositions for alleviating disorders associated with pneumococcal infection. For example, known pneumococcal vaccines comprise CPS obtained from numerous pneumococcal variants.
There remains a significant need for diagnostic, prognostic, therapeutic, and preventive compositions and methods useful with disorders associated with pneumococcal infection. Many of these compositions and methods comprise, employ, or rely for their development on isolated pneumococcal CPS. Previously known methods for isolating CPS from pneumococci are generally characterized by low yield. The present invention includes a method of improving the yield of CPS that can be obtained from a pneumococcal variant, and enhances production and use of diagnostic, prognostic, therapeutic, and preventive compositions and methods pertaining to pneumococcal infections.
The invention relates to an improvement in a method of producing capsular polysaccharide from a pneumococcus by maintaining the pneumococcus in a growth medium. In one aspect, the improvement comprises maintaining a gas having a sub-atmospheric concentration of oxygen in contact with the growth medium (e.g. an oxygen concentration not greater than about 16% or not greater than about 0.1%). The pneumococcus can be an organism of the genus Streptococcus, such as an organism of the species Streptococcus pneumoniae (e.g. one of S. pneumoniae variants 6A, 6B, 18C, and 9V). In another aspect, the improvement comprises maintaining a gas having a super-atmospheric concentration (e.g. at least about 3% or 10%) of carbon dioxide in contact with the growth medium. In a third aspect, the improvement comprises maintaining a gas having both a super-atmospheric concentration of carbon dioxide and a sub-atmospheric concentration of oxygen in contact with the growth medium. In still another aspect, the improvement comprises maintaining the carbon dioxide concentration of the growth medium at a level at least equal to the concentration of carbon dioxide in the same growth medium equilibrated at the same temperature with a gas comprising 5% carbon dioxide.
The invention also relates to a method of alleviating a pneumococcal infection in an animal. This method comprises maintaining the animal (e.g. at least the lungs of the animal) in contact with a gas having a super-atmospheric concentration (e.g. 25%, 50%, or 100%) of oxygen. Examples of infections which can be alleviated in this method include pneumonia, bacteremia, sepsis, and meningitis.
The invention further relates to a method of making an immunogenic preparation for administration to an animal at risk for developing a pneumococcal infection. This method comprises maintaining pneumococcal cells in a growth medium having an oxygen content lower than the same medium equilibrated at the same temperature with normal air and isolating capsular polysaccharide produced by the cells from the cells. The isolated polysaccharide constitutes the immunogenic preparation.
The invention still further relates to a method of producing pneumococcal polysaccharide. This method comprises maintaining pneumococcal cells in a growth medium having an oxygen content lower than the same medium equilibrated at the same temperature with normal air, for example, a medium substantially devoid of oxygen. In one embodiment, the medium has a carbon dioxide content which is greater than the same medium equilibrated at the same temperature with normal air, for example, a medium saturated with carbon dioxide or a medium comprising a carbonate or bicarbonate salt.
The invention includes a method of assessing whether a test compound is useful for alleviating a pneumococcal infection in an animal. This method comprises comparing
(a) the degree of phosphorylation of CpsD in pneumococcal cells maintained in the presence of the test compound and
(b) the degree of phosphorylation of CpsD in the same type of cells maintained in the absence of the test compound.
If the degree of phosphorylation of CpsD in the cells maintained in the presence of the test compound is less than the degree of phosphorylation of CpsD in the cells maintained in the absence of the test compound, then the test compound is useful for alleviating the infection. The degree of phosphorylation of CpsD can, for example, be assessed by assessing the number of phosphorylated tyrosine residues present in CpsD or by assessing the fraction of CpsD having at least one phosphorylated tyrosine residue.